Aldosterone-regulated sodium transport in the kidney distal nephron is essential for fluid and electrolyte homeostasis and blood pressure regulation in mammals. Aldosterone acts through the mineralocorticoid receptor (MR) to control transcription rates of a group of target genes, which have direct and indirect effects on key ion transporters, such as the epithelial sodium channel (ENaC). We recently identified the scaffolding protein, GILZ1 (glucocorticoid-induced leucine zipper protein, isoform 1), as an aldosterone-regulated gene product, which acts within distal nephron cells to stimulate ENaC cell surface expression. Our published and unpublished data demonstrate that GILZ1 interacts with ENaC (alpha and beta subunits), and a group of proteins already known to be involved in ENaC regulation. These ENaC-regulating GILZ-interacting proteins (GILZIPs) include SGK1 (a phosphatidyl inositol-3-kinase-dependent kinase, which stimulates ENaC), Nedd4-2 (a ubiquitin ligase, which inhibits ENaC), and Raf-1 (the master regulator of the Raf-MEK-ERK signaling module, which has effects on a wide array of cellular processes, including inhibiting ENaC). Based on these and other data, we suggest the novel hypothesis that these proteins are assembled into an ENaC regulatory complex (ERC), the composition of which is modulated by aldosterone, in a GILZ1-dependent fashion. In order to explore this general hypothesis, we propose the following specific aims: 1) Determine the molecular identity of the components of the ENaC regulatory complex (ERC), and the role of GILZ in controlling its composition. GILZ1 interacts with ENaC as well as with several ENaC regulatory proteins (which we have termed GILZIPs). We hypothesize that a multi-protein ENaC regulatory complex (ERC) is formed, and that GILZ1 is a key component of this complex, and recruits activating proteins into the complex. We will use biochemical and yeast two-hybrid approaches to examine this hypothesis, and determine the role of GILZ in ERC composition over time in response to aldosterone. 2) Determine the functional effects of GILZ1 and GILZIPs on endogenous ENaC-mediated Na+ currents in a cortical collecting duct (CCD) cell line, using gene specific knockdown and over-expression. 3) Determine the effect of GILZ1 and GILZIPs on ENaC surface expression and processing in cultured cells. We will examine the effects of GILZ1 and GILZIPs (particularly SGK1, Nedd4- 2 and Raf-1) on ENaC surface expression using biotinylation in cultured kidney epithelial cells; examine the effects of GILZ1 and GILZIPs on ENaC proteolytic processing; examine the effect of GILZ1 on processing of endogenous ENaC in mpkCCD cells. This work will elucidate the mechanistic basis of ENaC regulation by aldosterone, a process of fundamental importance to the regulation of BP and ECF volume. Moreover, it will shed new light on how specific regulation of ion transport can be achieved without unleashing undesirable effects on other processes, which is a central question in cell biology. PUBLIC HEALTH RELVANCE: The blood pressure-regulating hormone aldosterone is involved in controlling salt retention by the kidney. Abnormalities of this hormone are a major cause of hypertension, and are implicated in fluid overload states such as congestive heart failure. This proposal examines the molecules involved in mediating aldosterone signaling, and how it causes salt retention in the kidney. This work will shed new light on the basic mechanisms, and provide avenues for better treatment of high blood pressure and edema.